Everyone, I have a problem in my band. It occurs at high MW about 125 kDA in this picture while a band in low MW (not have a smear band). The protein is a phospho form. I lyse protein in NP-40 buffer with protease and phosphatase inhibitor and then centrifuge at 12000 rpm at 4 C. I run 10%gel at 90 v about 1.30 h and transfers on ice with 100v about 2 h. Blocking with 5%BSA in TBST and 1 AB 1:3000

Whether B-actin is ok.

I saw that smear band look like V-shape.

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